Ultrastructure and phylogenetic significance of the head ... · Ultrastructure and phylogenetic...

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Ultrastructure and phylogenetic significance of the head kidneys in Thalassema thalassemum (Echiura, Thalassematinae) Chiharu Kato 1 , Janina Lehrke 2 , Björn Quast 3 1, 2, 3 Rheinische Friedrich-Wilhelms-Universität Bonn, Institut für Evolutionsbiologie und Ökologie, An der Immenburg 1, D-53121 Bonn 1 Author to correspond to: [email protected] Tel.: ++49 228 73 57 58 Fax.: ++49 228 73 51 29 2 [email protected] 3 [email protected] Keywords: Head kidneys; Ultrastructure; Echiura; Annelida; Phylogeny; Larvae; Protonephridia Abstract Recent molecular analyses consistently resolve the "spoon worms" (Echiura) as a subgroup of the Annelida, but their closest relatives among annelids still remain unclear. Since the adult morphology of echiurans yields limited insight about their ancestry, we focused on characters of their larval anatomy to contribute to this discussion. Electron microscopical studies of the larval protonephridia (so called head kidneys) of the echiuran species Thalassema thalassemum revealed distinct correspondences to character states in serpulid polychaetes although a close relationship of Echiura and Serpulidae is not supported by any phylogenetic analysis. The larval head kidneys of T. thalassemum consist of only two cells, a terminal cell and a duct cell. The terminal cell forms a tuft of six cilia projecting 5 10 15 20 25

Transcript of Ultrastructure and phylogenetic significance of the head ... · Ultrastructure and phylogenetic...

Page 1: Ultrastructure and phylogenetic significance of the head ... · Ultrastructure and phylogenetic significance of the head kidneys in Thalassema thalassemum (Echiura, Thalassematinae)

Ultrastructure and phylogenetic significance of the head

kidneys in Thalassema thalassemum (Echiura,

Thalassematinae)

Chiharu Kato1, Janina Lehrke2, Björn Quast3

1, 2, 3 Rheinische Friedrich-Wilhelms-Universität Bonn, Institut für Evolutionsbiologie und Ökologie, An der Immenburg 1, D-53121 Bonn

1 Author to correspond to:[email protected].: ++49 228 73 57 58

Fax.: ++49 228 73 51 29

2 [email protected]

3 [email protected]

Keywords: Head kidneys; Ultrastructure; Echiura; Annelida; Phylogeny; Larvae;

Protonephridia

Abstract

Recent molecular analyses consistently resolve the "spoon worms" (Echiura) as a subgroup

of the Annelida, but their closest relatives among annelids still remain unclear. Since the

adult morphology of echiurans yields limited insight about their ancestry, we focused on

characters of their larval anatomy to contribute to this discussion. Electron microscopical

studies of the larval protonephridia (so called head kidneys) of the echiuran species

Thalassema thalassemum revealed distinct correspondences to character states in serpulid

polychaetes although a close relationship of Echiura and Serpulidae is not supported by

any phylogenetic analysis. The larval head kidneys of T. thalassemum consist of only two

cells, a terminal cell and a duct cell. The terminal cell forms a tuft of six cilia projecting

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into the lumen of the terminal cell. The cilia are devoid of circumciliary microvilli. A filter

structure is formed by two to three layers of elongate microvilli which surround the lumen

of the terminal cell in a tubular manner. A thin layer of extracellular matrix (ECM)

encloses the outer microvilli of the tubular structure. The tips of the microvilli project into

the lumen of the adjacent duct cell but are not directly connected to it. However, mechanic

coupling is faciliated by the surrounding ECM and abundant hemidesmosomes. The distal

end of the multiciliary duct cell forms the external opening of the nephridium; a

specialized nephropore cell is absent. Apart from the multiciliarity of the duct cell, details

of the head kidneys in T. thalassemum reveal no support for the current assumption that

Echiura is closely related to Capitellida and/or Terebelliformia. Available data for other

echiuran species, however, suggest that the head kidneys of T. thalassemum show a derived

state within Echiura.

Introduction

Echiura share numerous developmental and morphological characters with Annelida, like

the ultrastructure of chaetae and cuticle, the structure and position of the blood vessels, and

the development of the trunk mesoderm (Orrhage 1971; Ax 1999; Ruppert et al. 2004). In

addition, serially repeated ganglia in echiuran larvae correspond to typical metamerical

ganglia in annelids (Hessling and Westheide 2002; Hessling 2002; 2003). On the other

hand characters like the unsegmented trunk with a single secondary body cavity, the

extensible elongate muscular proboscis and the presence of anal sacs (anal vesicles sensu

Newby 1940) clearly distinguishes echiurans from typical annelidan taxa (Ax 1999;

Ruppert et al. 2004). Therefore, based on morphological data, Echiura were considered

either as a highly derived subtaxon of the Annelida (Eibye-Jacobsen and Nielsen 1996; Ax

1999; Nielsen 2001) or as separate taxon with close a relationship to annelids (Newby

1940; Korn 1960; Rouse and Fauchald 1995; Edmonds 2000). The former hypothesis

implies that Echiura have secondarily lost characteristic features of annelids like trunk

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segmentation, parapodia, and a metamerical nervous system in adults (Purschke et al.

2000; Bleidorn 2007).

Recent molecular and combined morphological and molecular analyses support a

systematic positioning of the Echiura within a clade comprising the annelid families

Terebellidae, Arenicolidae, Maldanidae and Capitellidae (Struck et al. 2007, 2008; Zrzavy

et al. 2009). Most of these analyses revealed a sister group relationship between Echiura

and Capitellidae but some multigene analyses also support a close relationship to

Terebellidae (Colgan et al. 2006) or Pectinariidae (Rousset et al. 2007, “restricted”

dataset). Morphological support for any of these sister group relationships is still wanting.

This is due to the problem that morphological characters traditionally used in echiurid

systematics are mostly lacking in other annelid taxa or show derived states within Echiura

(see Purschke et al. 2000; Ruppert et al. 2004).

Due to striking similarities in cleavage and early ontogenetic patterns it appears promising

to focus on morphological features of larval stages for phylogenetic inferences. Like most

polychaetous annelids, Echiura show a biphasic life cycle with a planktonic trochophore

larva (Baltzer 1931). It is generally accepted that this larva already evolved in the common

ancestor of the Trochozoa, which comprise at least Annelida, Entoprocta and Mollusca

(Rouse and Fauchald 1995; Ax 1999; Nielsen 2004, 2005). Trochophore larvae are

characterized by a specialized circumlarval ciliary belt (the prototroch), a sensory apical

organ, and one pair of transitory protonephridia (Rouse 1999; Nielsen 2004). These

protonephridia are located in the periphery of the larval blastocoel anteriorly to the anlagen

of the trunk mesoderm (Hatschek 1880; Goodrich 1945). Due to their position in the

presumptive head region they have been named head kidneys (“Kopfnieren”) by Hatschek

(1878, 1880). During metamorphosis the head kidneys disintegrate and become

functionally replaced by segmentally arranged nephridia in annelids or by the anal sacs in

most echiurans (Baltzer 1931; Goodrich 1945; Bartolomaeus and Ax 1992).

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Within Annelida the head kidneys of about 17 species have been investigated

ultrastructurally (see Bartolomaeus 1995; Bartolomaeus and Quast 2005). The structural

data of the head kidneys in these species provide a number of discrete characters, like their

composition (number of cells), the construction of the filtration area, and the ciliation in

the distinct nephridial parts and cells, respectively. Some of these features are characteristic

for high ranking subtaxa within the Annelida (Bartolomaeus 1995, 1998; Quast 2007) and

thus are useful for unraveling annelidan phylogeny.

A comparison of the head kidney morphology in Echiura could accordingly contribute

further insight on their phylogenetic position within Annelida. For this purpose, we

examined the head kidneys of Thalassema thalassemum (Pallas, 1766) (Echiuridae,

Thalassematinae) by means of transmission electron microscopy, providing the first

ultrastructural data on head kidneys in echiurans. The main goal of our study is to search

for structural correspondences that support a close relationship to the traditional Capitellida

(i.e. Capitellidae, Arenicolidae, and Maldanidae) as suggested by the above mentioned

phylogenetic analyses (Struck et al. 2007; Zrzavy et al. 2009).

Material and Methods

Reproductive adults of Thalassema thalassemum (Pallas, 1766) were collected in April and

May 2008 in Le Cabellou, Concarneau, France. Animals were taken from rock crevices in

the mid-intertidal zone. Adults were kept in small aquaria at the Freie Universität Berlin

with running artificial seawater (13–15°C).

Gametes where obtained by dissecting gonoducts containing ripe ova or sperm

respectively. Artificial fertilisation was conducted in glass bowls with ultrafiltrated cooled

seawater (9°C) from the Atlantic coast in Concarneau. The larvae were reared in a large

2 l beaker at 15–18°C. 98h old larvae were fixed for 1hin cold (4°C) 1.25%

glutaraldehyde buffered in filtered (0.2 µm) PBS (0.05 M sodium phosphate with 0.3M

NaCl, pH 7.2) containing traces of ruthenium red. After fixation, the larvae were washed

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three times in PBSand postfixed in 1% OsO4 in PBS for 1 h at 4° C. Subsequently, they

were dehydrated in a graded acetone series and embedded in araldite M (FLUKA).

Complete series of silver interference colored ultrathin sections (60–70 nm) were cut with

a diamond knife on a Leica Ultracut S microtome. The sections were mounted on formvar-

covered single-slot copper grids, automatically stained with uranyl-acetate and lead-citrate

with a Nanofilm TEM Stainer and examined with a Philips CM120 Bio-TWIN electron

microscope. Images were digitally recorded on imaging plates (DITABIS). The obtained

16 bit images were enhanced in contrast and converted to 8 bit gray levels with AnalySIS

(SIS Münster) or the the ImageJ software package (http://rsbweb.nih.gov/ij/). An aligned

series of selected micrographs of one head kidney is available at:

https://www.morphdbase.de?B_Quast_20110303-S-2.1. The 3D reconstruction of the

protonephridium was conducted using Adobe Illustrator CS 3 for segmentation of the cell

contours and Blender (http://www.blender.org) with the MorphMesh Plugin (http://www.q-

terra.de/biowelt/3drekon/index.html) for generating a 3D surface model. From the 3D

reconstruction virtual sections where imported into Adobe Illustrator and used as draft for a

schematic illustration of the protonephridium.

Results

The 98h old larva of T. thalassemum possesses one pair of protonephridia (Fig. 1). Each

protonephridium is tubular in shape and measures about 44 µm in length (Fig. 2). Both

nephridia are composed of two cells only, a terminal cell and a duct cell. They extend

straightly from the forgut anlage toward the anus and open to the exterior by piercing the

epidermis close to the anus. The external opening is situated ventrolaterally close to the

gastrotroch and ca. 40 µm anteriorly to the anus (Fig. 1). A specialized nephridiopore cell

that is embedded with most of its cell body into the epidermis is lacking.

Terminal cell

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The terminal cell is situated laterally to the anlage of the foregut and the mouth opening.

The cell is elongate and measures about 16 µm in length (Fig. 2). The proximal end of the

cell tapers off into a small apex. At this apex the terminal cell is connected to a muscle cell

via dense plaques (Fig. 3A, B). The nucleus is located within the proximal part of the

terminal cell, in a distance of about 2 µm to the apex. It contains a nucleolus and

heterochromatin. The nucleus has a diameter of about 3 µm.

With its distal most part the terminal cell forms a hollow, cylinder like compartment (Fig

2). The extracellular space represents the lumen of the terminal cell and is continuous with

the lumen of the adjacent duct cell. The outer wall of the cylinder is formed by numerous

elongate microvilli. The microvilli are about 3µm in length and emanate from the margin

of a small flattened area that has a diameter of about 1.5µm (Fig 3C). Actin filaments are

present within the microvilli (Fig 3D, E). In sections of their basal most part some

microvilli appear to be interconnected by cytoplasm (Fig 3D), but this is due to the level of

sectioning. No anastomoses or interconnections are found between the microvilli. The

microvilli are arranged in a ring-like area of about 0.3 µm thickness. Usually two to three

microvilli are orientated one behind the other suggesting an arrangement of at least two

irregular circles (Fig. 3D, 3F). The outer circle of microvilli is surrounded by a thin layer

of electron dense extracellular matrix. Occasionally, hemidesmosomes connect the

extracellular matrix to the underlying microvilli (Fig. 3D). The matrix is continuous with

the extracellular matrix surrounding the entire terminal cell. Any additional extracellular

membrane or diaphragm on or in between the microvilli seems to be absent.

From the flattened cytoplasmic area between the bases of the microvilli six cilia protrude

into the lumen of the terminal cell. Each cilium is anchored by a basal body with a short

rootlet and a lateral basal foot (Fig. 3C). The ciliary rootlet measures about 0.4 µm in

length. The rootlets of the six cilia are interconnected via microtubuli. None of the ciliary

basal structures possesses an accessory centriol.

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The cytoplasm of the terminal cell contains several mitochondria (Fig. 3A), smooth and

rough endoplasmatic reticula and golgi complexes. Several lipid droplets are located in the

perikaryon. In the region of the proximal apex and near the origin of the cilia, the

cytoplasm contains numerous coated and uncoated vesicles (Fig. 3C). Membrane pits

occur at the abluminal membran in the middle and distal part of the terminal cell.

Duct cell

The duct cell forms a stretched tubule with a length of approximately 30 µm (Fig. 2). The

nucleus is located in a lateral bulge in the middle part of the duct cell (Fig. 4D). It contains

heterochromatin and two nucleoli. Several golgi complexes, a dense system of

endoplasmatic reticulum and numerous mitochondria occur in the cytoplasm of the lateral

bulge. In the proximal part of the duct cell enlarged cisternae of the endoplasmatic

reticulum run almost parallel to the adluminal membrane and thus indicate a layer-like

construction of the adluminal cytoplasm (Fig 4A, B). Vesicles of various sizes are densely

distributed within the cytoplasm of the middle and distal sections of the duct cell (Fig 4D,

E). The adluminal and abluminal membranes of the duct cell possess numerous uncoated

membrane pits (Fig. 4B, C, D, E).

A thin electron dense layer of extracellular matrix surrounds the entire duct cell (Fig. 4A,

B). This basal membrane is continuous with the basal membrane of the terminal cell and

the adjacent epidermis cells. Hemidesmosomes connect the basal membrane to the duct

cell. The extracellular membrane in addition with hemidesmosomes solely provides the

mechanical connection between terminal and duct cell.

The lumen of the duct cell is percellular, i. e. the cytoplasm encompassing the lumen is

interconnected by an adluminal zonula adherentes and septate junctions (Fig. 4A, B, E). In

the proximal part of the duct cell the adluminal membrane forms neither cilia nor

microvilli. Only the cilia and microvilli of the terminal cell extend into this part of the duct

lumen (Fig. 4A, B). Because of their different length, the microvilli end successively at

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different levels in the proximalmost part of about 1 µm of the duct lumen. A direct

connection between the microvilli and the duct cell was not detected at any site of the

adluminal membrane. The six cilia of the terminal cell project about a length of 15 µm into

the lumen of the duct cell and thus extend to the level of its perikaryon (Fig. 4D). The duct

lumen of the proximal part of the cell has a diameter of about 0.5 µm (Fig 4B).

In the region of the perikaryon, the duct lumen widens to a diameter of 2.5 µm (Fig. 4D,

E). Here, about 15 cilia project from the adluminal membrane into the lumen. Each cilium

is anchored to the cytoplasm by a basal body with a lateral basal foot and a single rootlet.

The cilia insert separately and their basal structures are distributed over the whole

adluminal membrane in the middle part of the duct cell (Fig. 4D, E).

From the region of the nucleus on, there also protrude numerous finger-like cytoplasmic

processes into the nephridial lumen (Fig. 4D, E). This cytoplasmic protrusions

occassionally contain electron dense material, but no actin filaments were found within

them.

No cilia are originating from the distal part of the duct cell, but similar finger like cell

processes are observed as in the region of the perikaryon. The distalmost part of the duct

cell passes through the epidermis and forms the external opening of the nephridium (Fig

4F). In this region, the thickness of the cytoplasm encompassing the lumen thins out and

measures less than 0.2 µm. At its distal margin, the duct cell is connected to the adjacent

epidermis cells by adherens junctions. The nephridiopore is about 1 µm in diameter.

Fingerlike cell processes protrude from the duct cell at the margin of the nephridiopore and

partly overlap it (Fig. 4F). Some of the cilia of the middle part of the duct cell extend

through the nephridiopore into the external medium. A cuticular lining is absent both on

the exterior surface of the duct cell, as well as on the adjacent epidermis cells.

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Discussion

Based on a comparison of protonephridial systems in bilaterian animals, Bartolomaeus and

Ax (1992) hypothesized one pair of protonephridia for the ground pattern of the Bilateria.

In Annelida this pair of protonephridia exists as head kidneys exclusively in early

developmental stages and is replaced by serially repeated nephridia in the trunk segments

during postembryonic or postlarval development. The segmentally arranged nephridia are

either protonephridia or metanephridial systems (Goodrich 1945; Ruppert and Smith 1988;

Bartolomaeus 1999). Head kidneys and segmental protonephridia of Annelida often exhibit

an increase of the number of circumciliary microvilli in the terminal cell to at least ten

(Bartolomaeus and Ax 1992; Bartolomaeus 1995). According to their comparative survey

of protonephridia in Bilateria Bartolomaeus and Ax (1992) assume that protonephridia

composed of three cells only, one terminal cell, one duct cell and one nephropore cell

belong to the ground pattern of Bilateria.

Structural variability of head kidneys within Annelida

Within Annelida head kidneys consisting of three cells have hitherto only been found in

Chaetopterus variopedatus (Renier, 1804), Spirorbis spirorbis (Linnaeus, 1758),

Magelona mirabilis Müller, 1858, and Scoloplos cf. armiger (Bonch-Bruevich and

Malakhov 1987; Bartolomaeus 1993a, 1995, 1998). In terebelliform species investigated

thus far the head kidneys are composed of two terminal and duct cells each, while

“oligochaetous” clitellates show a reduction of the nephridiopore cell (Bartolomaeus 1995;

Quast 2007). Due to the lack of ultrastructural data currently no further apomorphy

hypotheses can be drawn from the cell numbers in annelidan head kidneys (Bartolomaeus

1995; Bartolomaeus and Quast 2005, see below). In contrast ultrastructural data of the

terminal cells and their filtration sites provide additional character states with phylogenetic

significance. In species of the Phyllodocidae and Syllidae it was shown that the

cytoplasmic cylinder of the terminal cell is reduced; the supporting structure of the filter is

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solely formed by strengthened circumciliary microvilli (Bartolomaeus 1989, 1993b). The

same structural peculiarity proved to be shared by taxa closely related to Syllidae and

Phyllodocidae and was accordingly considered as an autapomorphy of a larger subtaxon

within the Phyllodocida comprising at least Phyllodocidae, Syllidae, Alciopidae,

Pisionidae, Nephtyidae Glyceridae, Tomopteridae, Hesionidae and Pisionidae

(Bartolomaeus 1995, 1997). A supporting structure of the filtration matrix formed by

cylindrically arranged microvilli, as now revealed for the echiuran T. thalassemum, has

never been described for any head kidneys in annelids. The filter-forming terminal cell of

the head kidneys of this echiuran, however, corresponds to the state in two serpulid

species, Serpula vermicularis Linnaeus, 1767 and Spirorbis spirorbis, in bearing a tuft of

several cilia (Pemerl 1965; Bartolomaeus 1993a). In the serpulid species, eachcilium of the

tuft is devoid of circumciliary microvilli, but a huge number of microvilli surround the

entire ciliary tuft as in T thalassemum. Serpulids differ, however, from T. thalassemum

since a slashed outer cytoplasmic cylinder encloses the microvilli (Table 1).

Further correspondences with these two serpulid species unfortunately remain unclear as

no detailed data are available for the number of cells forming the duct and the nephropore.

Among polychaetes, however, the lack of a nephropore cell as revealed for T. thalassemum

is also only known thus far from a serpulid species, Pomatoceros triqueter (Linnaeus,

1767) (Wessing and Polenz 1974) (Table 1). This correspondence is furthermore shared by

the "oligochaete" taxa Tubifex sp. and Dendrobaena veneta (Rosa, 1886) among clitellates

(Quast 2007). These two “oligochaete” clitellates differ from T. thalassemum in that the

single terminal cell of the head kidneys is transformed into a nephrostome that opens into a

secondary body cavity. Among polychaetes, head kidneys connected to a secondary body

cavity by a proximally opened nephrostome only occur in spionid species (Schlötzer-

Schrehardt 1992; Bartolomaeus and Quast 2005). This transformation of the terminal cell

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has accordingly been considered as potential synapomorphy of Spionida and Clitellata

(Quast 2007).

The head kidneys of T. thalassemum presently reveal no distinct morphological support for

a close affinity of Echiura to a clade of Capitellidae, Maldanidae, Arenicolidae, and

terebelliform taxa, as has been retrieved in recent molecular analyses (e.g. Bleidorn 2003a,

2003b; Hall et al. 2004; Colgan et al. 2006; Struck et al. 2007; Zrzavy et al. 2009).

Ultrastructural data on the head kidneys in these polychaetes are only available for the

terebelliform species Lanice conchilega (Pallas, 1766) (Terebellidae) and Pectinaria

auricoma Müller, 1776 (Pectinariidae), as well as some preliminary personal observations

for the capitellid species Capitella teleta Blake, Grassle and Eckelbarger, 2009. In these

species, the head kidneys are composed of two or more terminal and duct cells and are

equipped with a nephropore cell (Heimler 1981, 1983; Bartolomaeus 1995). The terminal

cells variably differ in the number and arrangement of cilia, which possess circumciliary

microvilli, as well as in their filtration structures that are basically formed by clefts in the

terminal cell's cytoplasm. The apparent absence of filtration structures in L. conchilega

caused Heimler (1988) to assume that the head kidneys are nonfunctional in this species,

but it cannot be excluded that in the developmental stages studied by him they already

started to disintegrate (Bartolomaeus 1995). The head kidneys of T. thalassemum and the

terebelliform and capitellid polychaetes studied thus far only seem to correspond in the

multiciliarity of the duct cells. Multiciliary duct cells are widespread in annelids but the

ancestral state in annelids (multi- versus monociliary duct cells) still remains unclear

(Bartolomaeus, pers. communication).

Stuctural variability of head kidneys within Echiura

For echiuran species other than T. thalassemum only light microscopic data on the head

kidneys are available. In the Bonelliidae, their structure basically seems to correspond to

the state in T. thalassemum. Baltzer (1914) described the head kidneys in late larvae of

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Bonellia sp. as unbranched tubes with a blind terminal end bearing solenocytes (i.e.

terminal cells with a single cilium surrounded by an elongate tubular filtration structure;

see Goodrich 1898, p. 442). Dawydoff's (1959) illustration of a head kidney instead

suggests that the terminal end forms a multiciliary tuft. More detailed information on the

terminal structure and on the number of cells composing the head kidneys in bonellids is

still missing.

Head kidneys in the Echiurinae, in contrast, seem to show surprising similarities to the

state in C. teleta (Capitellidae). In the larva of Echiurus sp. the head kidneys were

described as short branched tubular ducts with several monociliary terminal cells

connected to the ducts via a thin pipe-like tubule (Hatschek 1880; Goodrich 1910; Baltzer

1917; Korn 1960). Strikingly, the definitive protonephridia in the dwarf males of B. viridis

(Bonellidae) show a similarity to to the head kidneys in Echiurus larvae since they possess

several terminal cells. Based on ultrastructural studies, Schuchert (1990) found

protonephridia in males of B. viridis composed of five terminal cells that jointly attach to

the terminal end of an unbranched multicellular duct. As in T. thalassemum, the terminal

cells form ciliary tufts surrounded by numerous microvilli-like cell protrusions, but they

differ from the state in T. thalassemum in the higher number of about 20 cilia and in the

presence of anastomotic interconnections between the microvilli-like cell protrusions.

Because these protonephridia arise in postlarval stages and are differently positioned at the

posterior end of the trunk, the protonephridia in males of B. viridis are not homologue to

the larval head kidneys in other species and hence could be unsuitable for any phylogenetic

inference. The structural similarities of the head kidneys and segmental protonephridia in

polychaetes like Polygordius sp., Glycera dibranchiata Ehlers, 1868, and Phyllodoce

("Anaitides") mucosa Oersted, 1843, however, support the view that they are serially

homologous and thus probably based on the same genetic information (Smith and Ruppert

1988; Bartolomaeus 1989). Since the interrelationships among the echiuran high-ranking

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subtaxa still remain unclear (see, e.g., Lehrke and Bartolomaeus 2009), the

correspondences of the protonephridia in Echiurus larvae (Echiurinae), the dwarf males of

B. viridis (Bonellidae), and larvae of C. teleta (Capitellidae) may accordingly indicate that

the head kidneys in larvae of T. thalassemum (Thalassematinae) and Bonellidae are derived

from the state shown by Echiurus larvae.Conclusions

Recent molecular insight on a close relationship between Echiura and the polychaete

subgroups Capitellida and Terebelliformia receives no corroboration by the structure of the

larval head kidneys in T. thalassemum. In this echiuran species, the head kidneys rather

correspond to states in serpulid polychaetes. Available light microscopical data on

Echiurus and preliminary data on Capitella larvae, however, indicate that this resemblance

in Thalassema and Serpulidae might rather be based on convergent transformations.

Further comparative ultrastructural studies of the head kidneys based on a denser taxon

sampling are promising to clarify their evolution.

Acknowledgements

We specially thank Markus Koch for valuable comments and discussions that improved the

manuscript. We are also grateful to Thomas Bartolomaeus and Jörn von Döhren for support

with rearing and fixation of T. thalassemum larvae. Two anonymous reviewers are

acknowledged for helpful comments on the manuscript.

References

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Fig. 1 Schematic drawing of the 98h old larva of Thalassema thalassemum, lateral view.

The head kidneys (hk) are located ventrolaterally beside the stomach (st) and the intestinal

pouch (ip) (only left head kidney is shown). Each head kidney extends from the level of

the mouth opening (mo) towards the posterior third of the larva, where the nephropore (np)

is located. an anus; fg foregut anlage; gt gastrotroch; mt metatroch; pt prototroch; tt

telotroch.

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Fig. 2 Schematic representation of the head kidney in the

98 h old larva. The head kidney is composed of two cells

only. The filtration structure is formed by elongated

microvilli (mv) emerging from the terminal cell (tc). ci

cilium; dc duct cell; mc muscle cell, nu nucleus; ro ciliary

rootlet; tc terminal cell.

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Fig. 3 TEM, cross sections of the terminal cell a The apex of the cell (tc) is connected to a

muscle cell (mc) via dense plaques (arrowhead). b The nucleus (nu) is located in the

proximal part of the terminal cell (tc). The perikayon shows further connections to the

muscle cell (mc) by dense plaques (arrowhead). c The distal end of the cell forms a

flattened area from which six cilia protrude into the nephridial lumen. Basal bodies (bb)

with a short rootlet and a basal footlet (arrow) anchor the cilia within this area. d Distally

circularly arranged microvilli (mv) enclose the lumen (lu) of the terminal cell. The

outermost microvilli are covered by a thin layer of extracellular matrix (ecm) to which they

are connected by hemidesmosomes (asterisk). e Actin filaments (af) are present within the

microvilli. f Transition from the terminal cell to the duct cell (dc). The thin layer of

extracellular matrix (ecm) that surrounds the outermost microvilli (mv) is continuous with

the matrix enclosing the duct cell (dc). ci cilium

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Figure 4 TEM, cross sections of the duct cell a Proximal part of the duct cell (dc). In this

section the duct cell shows a small lateral bulge (bl), that fuses with the rest of the cell in

the subsequent sections. Cilia (ci) and microvilli (mv) of the terminal cell extend into the

duct lumen. arrowhead indicates adluminal adherens junction. b Cross section of duct cell

proximally to the perikaryon.The lumen contains cilia (ci) of terminal cell. The duct cell

enfolds the percellular lumen. An adluminal adherens junction (arrowhead) forms a

longitudinal seal along its whole length. The adluminal membranes possess pits indicating

transcytotic processes (asterisk and inset c). d Middle part of the duct cell (dc) with

nucleus (nu). Lumen contains cilia (ci) and cytoplasmic protrusions originating from the

adluminal membrane of the duct cell. e Detail from d, showing finger-like cytoplasmatic

protrusions (cp) and pits at the adluminal membrane. The cytoplasm of the duct cell

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contains numerous vesicles (ve). f The distal part of the duct cell pierces the epidermis (ec)

and opens to the exterior via a nephropore. Some cilia (ci) extend through the nephopore to

the outside.

ecm electron dense extracellular matrix;er endoplasmatisches reticulum; lu lumen of duct;

mc muscle cell, mi mitochondrium.

510

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Table 1: Character states of head kidneys in Echiura and outgroup taxa supposed to be closely related to Echiura by Struck et al. (2007) and Zrzavy et al. (2009)

Species Terminal structure Duct Nephridiopore References

# cells # cilia / cell cmv filter # cells # cilia / cell mv # cells # cilia / cell

EchiuraThalassema thalassemum

1mc (6)

- mv ring 1 ~15 - - n.a. this paper

Echiurus sp.1 several1

? ?, * ? ? ? ? ? Hatschek 1880; Goodrich 1910

Bonellia sp.1 ? mc - ? ? ? ? ? ? Baltzer 1931

Bonellia viridis2 dwarf male posterior protonephridium

5mc

- perforated cytoplasm

many mc - ? ? Schuchert 1990

CapitellidaCapitella teleta several

1-210-11 perforated

cytoplasm>2 mc ? 1 ? pers. observation

TerbelliformiaLanice conchilega 2

mc (~15)? ? 2 ? ? 1 ? Heimler 1981, 1983,

1988Pectinaria auricoma 2

mc (~30)8-12 perforated

cytoplasm2 mc ∞ 1 - Bartolomaeus 1995

ClitellataDendrobaena veneta 1 mc (∞) - nephrostome 1 mc ∞ - n.a. Quast 2007Tubifex sp. 1 mc (∞) - nephrostome 1 mc ∞ - n.a. Quast 2007

SerpulidaPomatoceros triqueter 1

mc- “Weir” mc ? - n.a. Wessing & Polenz

1974Serpula vermiculosus 1

mc- perforated

cytoplasm? mc ? ? ? Pemerl 1965

Spirorbis spirorbis 1 mc - perforated cytoplasm

1 mc ∞ 1 mc Bartolomaeus 1995; Bartolomaeus & Quast 2005

All data refer to ultrastructural investigations except for taxa marked with1 (light microscopic data); 2 data from definitive nephridium; * “Solenocyte” according to Goodrich 1910; cmv circumciliary microvilli; mc multiciliated; mv microvilli; n.a. not applicable515